1. Field of the Invention
The invention concerns a method for determining antithrombin III (AT) in body fluids by adding an AT binding partner to the sample and determining the free AT binding partner. It also concerns a reagent that is suitable for this method.
2. Description of Related Art
AT is a factor of the blood coagulation system which plays a regulatory role. Blood coagulation is initiated by a cascade-like interaction of various proteases. The last of the successive activation steps releases thrombin, which in turn generates fibrin monomers which associate to form a thrombus. The most important regulator is AT, which can form a complex with thrombin and also with other proteases involved in blood coagulation that blocks the active center. The AT content in the blood of healthy humans is within a relatively narrow range. Reduced AT contents may be due to consumptive coagulopathy, a severe liver disease or they may be hereditary. A reduced AT content is nowadays generally regarded to be a risk for thrombosis. Hence in some cases the AT content is even reduced in an acute thrombosis. Therefore the AT content is a valuable parameter in clinical diagnostics.
Various methods are already known for detecting AT in which an AT binding partner is added to a sample under conditions that allow an interaction of the AT binding partner with AT present in the sample and subsequently determining the amount of free AT partner. Such determinations may, for example, be based on immunological methods or use chromogenic substrates. In the latter case, thrombin or activated factor X is, for example, added to the sample which interacts with the AT present in the sample. Excess thrombin is then determined by incubation with a chromogenic substrate which forms a coloured substance due to the action of thrombin and evaluation of the colour generation where the AT content is indirectly proportional to the colour formation. Methods for determining AT are described, for example, in Bergmeyer, Methods of Enzymatic Analysis, 3rd edition, “Verlag Chemie”, vol. 5, p. 441-448; I. Witt, ed., “Neue Methoden der Gerinnungsanalyse mit chromogenen Substraten”, Stormorken, “Neue Methoden der Gerinnungsanalyse”, page 119-121; Odegard et al., Haemostasis 7: 202-209 (1978); Fareed et al., Chromogenic Peptide Substrates (eds. M. F. Scully and V. V. Kakkar) Churchill Livingstone (1979) 183-191 and Abildgaard et al., Thromb. Res. 11, 549-553 (1977).
A disadvantage of known methods for detecting AT by adding thrombin is that a false high AT value is obtained in the presence of interfering factors, e.g., drugs such as hirudin that can themselves interact with thrombin. This disadvantage can be avoided by using activated factor Xa instead of thrombin. However, at present several factor Xa inhibitors are under development as therapeutic agents (Ostrem et al., Biochemistry 37 (1998), 1053-1059; U.S. Pat. Nos. 5,783,421; 5,721,214; WO 96/40679; U.S. Pat. No. 5,693,641; WO 97/46523; JP-96-191434 etc.). When these agents come onto the market, the same problems will occur with a factor Xa-based detection method as with the thrombin-based test.
Hence the object of the invention was to improve known detection methods and to provide a method that leads to a reliable measured result even in the presence of interfering factors in the sample to be tested.